The extracellular adenosine receptors have a modulatory role in the nervous, circulatory, endocrine and immunological systems. The prospect of harnessing these effects specifically for therapeutic purposes is attractive. We have synthesized the first selective A3 adenosine receptor agonists and antagonists. We recently characterized the first allosteric modulators of this receptor. Imidazoquinoline and pyridinylisoquinoline derivatives were found to enhance the actions of agonists of the A3 receptor, and thus may prove to be suitable leads for the development of therapeutic agents based on this concept. A3 agonists are under development for treating cancer, rheumatoid arthritis, and other diseases. Allosteric modulators of G-protein coupled receptors are a new approach to receptor activation without the limitations of use of directly-acting agonists as therapeutic agents. We have identified two new classes of allosteric modulators of the A3 receptor and are currently exploring the structure activity relationship (SAR).[unreadable] [unreadable] The potential of A3 agonist therapy is of great interest. We are collaborating with Dr. Bruce Liang and Dr. Asher Shainberg on various aspects of the use of adenosine receptor agonists in protection of the heart. We have designed a mixed agonist of A1 and A3 subtypes, both of which are protective in the heart. This mixed agonist MRS3630 is protective in a model of ischemia in the isolated perfused mouse heart. The adenosine A3 agonist IB-MECA is currently in clinical trials for use in cancer treatment by our CRADA partner Can-Fite Biopharma. [unreadable] [unreadable] Adenosine A3 antagonists may be useful for the treatment of glaucoma. Early efforts to identify antagonists of the A3 receptor in our library involved screening of chemically diverse libraries. One of the limitations of this approach is that the antagonists often bind well only at the human, but not murine A3 receptors. We are currently developing other novel A3 antagonists based on nucleotide structures, that have proven to be generally applicable across species. We are currently studying systematically the SAR of adenosine derivatives that affect efficacy as A3 adenosine receptor agonists. Surprisingly, a commonly used A1-selective agonist, cyclopentyladenosine, was found to act as a pure antagonist at the A3 subtype. Other nucleosides may be chemically modified, especially on the ribose moiety, to have reduced efficacy at the A3 receptor. Some of these analogues derived from highly potent A3 agonists, such as 5'-dimethylamides, were found to be A3 antagonists. A novel nucleoside-based antagonist of the A3 receptor MRS1292 was found to lower intraocular pressure a mouse model of glaucoma.[unreadable] [unreadable] We are using mutagenesis to study the determinants of recognition of adenosine within the binding site of the A2A and A3 receptors, and proposing conformational factors involved in receptor activation. Since the four subtypes of adenosine receptors have been cloned it has been possible to conduct molecular modeling of the receptor protein, based on sequence analyses and homology modeling using the high resolution rhodopsin structure as template. We intend to use such a modeling approach for the design of more selective adenosine receptor agonists and antagonists. [unreadable] [unreadable] Receptor engineering has also been explored with the design and expression of constitutively active A3 receptors (continuously activated, in the absence of agonist) and the introduction of the concept of neoceptors. Neoceptors are tailor-made mutant receptors designed to respond only to synthetic agonist derivatives that do not activate the native receptor, We have utilized molecular modeling of the human A3 receptor to demonstrate the feasibility of the neoceptor approach using a novel electrostatic interaction within the binding site created by modifying both the receptor and ligand in concert. The neoceptor approach has been extended to the A2A adenosine receptor, activation of which has an antiinflammatory effect. Recently this project has also focused on the effects of adenosine agonists and antagonists in the central nervous system and in the heart and on the possibility of therapeutics for treating neurodegenerative and cardiovascular diseases. An A3 agonist, administered chronically, proved to be highly cerebroprotective in an ischemic model in gerbils. A3 agonists cause morphological and biochemical changes in astroglial cells. Adenosine is released in large amounts during myocardial ischemia and is capable of activating both A1 or A3 receptors that occur on cardiac myocytes to exert a potent cardioprotective effect. We have shown that synthetic adenosine agonists, selective for either the A1 or A3 subtype, protect ischemic cardiac myocytes in culture and in the isolated perfused heart and thus might be beneficial to the survival of the ischemic heart. An acutely administered A3 agonist, Cl-IB-MECA, was cardioprotective in cell culture, through the selective activation of A3 receptors without side effects, such as bradycardia, associated with the A1 subtype. The protection was blocked in the presence of a selective A3 receptor antagonist. Inn summary, highly selective adenosine analogues may have therapeutic potential in treatment of cerebral ischemia/stroke and possibly other neurodegenerative disorders as well. It is proposed that modulation of A2B and A3 receptors may be useful in treating asthma and inflammatory diseases. The pharmacolgical properties of novel xanthines developed in our lab that act as selective A2B receptor antagonists are being explored as potential antidiabetic and antiasthamtic agents. The first highly selective A2B receptor antagonist, synthesized in our section, has now been radiolabeled and is used in assaying newly synthesized analogues for affinity at the A2B receptor. We are also screening chemical libraries for novel leads for A2B receptor antagonists.